筛选调节HBV增殖和HBsAg产生的宿主细胞的miRNA  

Screening host cell miRNA regulating HBV proliferation and HBsAg expression

作　　者：章广玲[1,2] 王梅梅[1] 朱丽华[1,2] 熊亚南[1] 李怡璇[2] 刘民[2] 汤华[2] 袁丽杰[1] 

机构地区：[1]河北联合大学基础医学院病原生物学教研室,河北唐山063000 [2]天津医科大学天津市生命科学中心实验室,天津300070

出　　处：《第三军医大学学报》2012年第1期44-48,共5页Journal of Third Military Medical University

基　　金：天津市自然科学基金(09JCZDJC17500)~~

摘　　要：目的筛选调节乙型肝炎病毒(HBV)增殖和乙型肝炎病毒表面抗原(HBsAg)产生的宿主细胞的miRNA。方法用328种miRNA的反义链转染HepG2 2.2.15细胞,72 h收获上清,MTS检测细胞的增殖活性,ELISA检测表面抗原和e抗原的表达水平,实时定量PCR检测其中HBV DNA的拷贝数。构建筛选到的miRNA的过表达载体,转染HepG22.2.15细胞,也分别用MTS、ELISA和实时定量PCR进行检测。结果 miR-199a-3pASO、miR-210ASO和miR-185ASO促进了HBsAg产生和HBV增殖(P<0.05);miR-199a-3p、miR-210、miR-185的过表达,分别抑制了HBsAg的产生和HBV的增殖(P<0.05);miR-199a-3p、miR-210和miR-185的ASO转染组与对照组相比,分别降低了细胞内miR-199a-3p、miR-210和miR-185的表达水平(P<0.05);miR-199a-3p、miR-210和miR-185的过表达载体转染组与对照组相比,分别增加了细胞内miR-199a-3p、miR-210和miR-185的表达水平(P<0.05)。结论在没有对细胞的增殖活性产生影响的情况下,miR-199a-3p、miR-210和miR-185抑制了HBV表面抗原的产生和HBV的增殖,miRNA作为病毒与宿主细胞相互作用中的调节因子,在HBV的生命活动周期中具有重要作用。Objective To screen out miRNAs that regulate HBsAg expression, HBeAg expression and proliferation of HBV in HepG2 2.2.15 cells, and to provide evidences for interaction between host cell＇ s miRNAs and HBV. Methods Three hundred and twenty eight antisense oligonucleotides of miRNAs were used to inhibit expressions of endogenous miRNAs. Enzyme linked immunosorbent assay （ELISA） and real- time PCR were performed on culture supernatant, which was harvested from HepG2 2.2.15 cells at 72 h after transfection with miRNA ASOs, to detect expressions of HBsAg and HBeAg and copy number of HBV DNA, respectively. Cell proliferation assay （MTS） was used at the same time. Results Three miRNAs, including miR-199a-3pASO, miR-210ASO and miR-185ASO, promoted HBsAg expression and HBV proliferation （P 〈 0.05）. Overexpression of miR-199a-3p, miR-210 and miR-185 inhibited HBsAg expression and HBV prolifer- ation （ P 〈 0.05 ）. Compared with those of the control group, HepG2 2.2.15 cells transfected with miR-199a- 3pASO, miR-210ASO and miR-185ASO showed a decreased level of miR-199a-3p, miR-210 and miR-185, respectively （P 〈 0.05）. Cells transfected with miR-199a-3p, miR-210 and miR-185 showed an increased levels of miR-199a-3p, miR-210 and miR-185 compared with those of the control group （P 〈0.05）. Conclu- sion miR-199a-3p, miR-210 and miR-185 inhibit HBsAg expression and HBV proliferation without inhibition of cell proliferation, miRNA, which regulates the interaction between host cell＇ s miRNAs and HBV replication, plays an important role in HBV proliferation.

关 键 词：MIRNA 乙型肝炎病毒 乙型肝炎病毒表面抗原 

分 类 号：R392-33[医药卫生—免疫学] R373.21[医药卫生—基础医学]